Pseudoxanthoma elasticum (PXE), a heritable disorder characterized by progressive calcification of elastic structures in the skin, eyes and the cardiovascular system, is caused by mutations in the ABCC6 gene, which encodes MRP6, a member of the ABC transporter family of proteins. Surprisingly, ABCC6 is primarily expressed in the liver and the kidneys, tissues not known to be involved in PXE, suggesting that PXE is a metabolic disease. The precise function of MRP6, consequences of the ABCC6 mutations at the mRNA and protein levels, and the pathomechanisms leading to calcification of the elastic structures are currently unknown. In this application, we propose multidisciplinary state-of-the-art approaches to dissect the pathomechanisms leading to the PXE phenotype based on the unifying hypothesis that PXE is a metabolic disorder of the genome/environment interface. Specific Aim 1 proposes development of animal models for PXE by ablation of the ABCC6 gene in global and liver-specific manner utilizing gene targeting by mouse ES cell technology. In fact, we have already developed an ABCC-/+ mouse which demonstrates ectopic mineralization in the skin, the retina, and the mid-sized arteries, thus recapitulating features of PXE. The transgenic animals will be examined further for phenotypic manifestations by clinical, histopathological, immunohistochemical, and electron microscopic means. Towards development of potential gene therapy for PXE, the ABCC6-/- mice will be used as a target of gene transfer of the ABCC6 gene under control of liver-specific promoter. Furthermore, we will examine the effects of syngenic liver transplantation and cell-based reconstitution on PXE phenotype in ABCC6 mice. Specific Aim 2 deals with elucidation of consequences of the ABCC6 mutations at the cellular and tissue level. First, to test the hypothesis that PXE is a primary metabolic disorder, we will subject plasma, urine, and bile from ABCC6-/- mice and plasma and urine from PXE patients for metabolic screen with emphasis on conjugated anionic organic compounds. An alternate hypothesis, viz. that the mutations lead to altered gene expression in the resident cells localized in the lesional tissues, will be tested by gene expression profiling of cells from PXE patients'skin as well as from ABCC6-/- mice, using microarray hybridization technology. Finally, towards identification of the endogenous substrate for MRP6, we will generate a transgenic mouse which traffics MRP6 to the apical, rather than normal baso-lateral membrane location, allowing analysis of bile as the source of the endogenous metabolite responsible for PXE phenotype.We anticipate that these approaches will disclose the pathomechanisms explaining the consequences of ABCC6 gene mutations at the phenotypic level. Such information is critical for development of translational strategies to counteract this devastating multi-system disease.